An inducible tricolor reporter mouse for simultaneous imaging of lysosomes, mitochondria and microtubules

Cell-type-specific use of the same DNA blueprint generates diverse cell types. Such diversity must also be executed via differential deployment of the same subcellular machinery. However, our understanding of the size, distribution, and dynamics of subcellular machinery in native tissues, and their connection to cellular diversity, remain limited. We generate and characterize an inducible tricolor reporter mouse, dubbed “kaleidoscope”, for simultaneous imaging of lysosomes, mitochondria and microtubules in any cell type and at a single cell resolution. The expected subcellular compartments are labeled in culture and in tissues with no impact on cellular and organismal viability. Quantitative and live imaging of the tricolor reporter captures cell-type-specific organelle features and kinetics in the lung, as well as their changes after Sendai virus infection. Yap/Taz mutant lung epithelial cells undergo accelerated lamellar body maturation, a subcellular manifestation of their molecular defects. A comprehensive toolbox of reporters for all subcellular structures is expected to transform our understanding of cell biology in tissues.


INTRODUCTION
Nearly all cells in our body have the same DNA and subcellular machinery, with few exceptions due to specialization such as somatic mutations for adaptive immunity and denucleation of red blood cells. Selective use of the same DNA blueprint underlies our ~200 cell types and must be executed via cell-type-specific use of the subcellular machinery, such as the T-tubules of muscles, microvilli of absorbent gut cells, synaptic junctions of neurons, and lysosomal related organelles of melanocytes. Besides such causation, a cell type is associated with and can be defined by not only expressed genes, but also cellular features, as exemplified in Ramon y Cajal's neuronal classification by cell morphology 1 . Moreover, the myriad DNAcoded macromolecules, as well as their cofactors, substrates and products, are confined to organelles, transported via the vesicular system along the cytoskeleton, and connected through cell-cell and cell-matrix adhesions.
Despite this analogy and interplay between molecular and cellular features, the former has enjoyed robust understanding and tools, leading to a conceptual framework where combinatorial action of transcription factors drives cell-type-specific epigenomic changes and consequently gene expression. In contrast, studies of cellular features are typically limited to cultured cells due to technical feasibility. However, as genes have distinct functions across cell types, subcellular machinery may also function differently in culture versus in tissues, particularly between large thin cells on 2D stiff plastics and those embedded in 3D cellular and matrix environment. Although classic transmission electron microscopy can pinpoint many subcellular structures, it is challenging to assign them to specific cell types, track them over time, and construct a 3D view for the entire cell.
The necessity to study cellular features in tissues and the difficulty in doing so are accentuated in the gas-exchanging lung alveoli, where a multitude of cell types interdigitate within thin tissues bordering millions of air pockets. For example, individual alveolar type 1 (AT1) cells and Cap2 endothelial cells (also known as Car4 cells or aCaps) span several hundred microns and multiple alveoli, whereas mesenchymal cells are as diverse in morphology and location [2][3][4][5][6] . Furthermore, their cellular processes are often too thin and intertwined to attribute subcellular staining signals to their cellular source. Inspired by the sparse cell labeling strategy achieved originally via Golgi staining and nowadays by genetic recombination 7 , we have visualized the diverse morphology of each cell type in the alveolar epithelial, endothelial, and mesenchymal lineages using a Cre-dependent cell membrane or cytosolic reporter 2,5,6 . Expanding this pipeline of cell biology analysis, our current study has generated a reporter mouse to fluorescently label subcellular structures including lysosomes, mitochondria, and microtubules. Unlike existing reporters that are limited to neurons or individual structures [8][9][10][11][12][13] , our reporter enables tricolor labeling within a single cell of any type for which a Cre driver is available. Applying this reporter across organs and cell lineages, using live imaging, in a viral injury model, and in conjunction with genetic mutations, we have shown in native tissues celltype-specific distributions of lysosomes and mitochondria, lysosomal fusion and fission, organelle changes upon viral infection, and lamellar body biogenesis and its defects in Yap/Taz mutant alveolar type 2 (AT2) cells.

Concatenated fluorescent fusion proteins label three subcellular structures when transiently expressed
Recognizing the use of ROSA reporters in not only lineage tracing, but also visualizing cell morphology (ROSA mTmG and ROSA tdT ) and purifying ribosomes (ROSA L10GFP ) or nuclei (ROSA Sun1GFP ) [14][15][16][17] , we sought to systematically label subcellular structures, leveraging myriad fluorescent fusion proteins verified in cultured cells, to fill the gap in our knowledge of cell biology in the native tissues. To achieve comparable expression among multiple fusion proteins, we concatenated them with 2A self-cleaving sequences, instead of IRES [18][19][20][21] , and positioned the largest and thus potentially less robust (tag)BFP-Lamp1 in the preferred first position, before a mitochondrial localization signal (MLS)-linked mKate2 (a tagRFP variant) and an EGFP-tagged alpha-Tubulin (Fig. 1A, S1A). We named this tri-fusion protein construct "kaleidoscope" given its ability of multicolor labeling of structures inside a cell. We placed the kaleidoscope construct under the control of a strong ubiquitous CAG promoter used in other ROSA reporters, separated by a Cre-dependent transcriptional stop cassette.
To test the kaleidoscope construct, we removed the transcriptional stop cassette in vitro with a Cre recombinase and transiently transfected the Cre-recombined construct into a HEK293T human cell line 22 . Native fluorescence from BFP-Lamp1 exhibited a few perinuclear globules, MLS-mKate2 a vesicular network, and EGFP-alpha-Tubulin a reticular pattern excluding the cell nucleus and BFP-Lamp1 globules -distributions consistent with lysosomes, mitochondria, and microtubules, respectively (Fig. 1B). A similar pattern was observed in a mouse lung epithelial cell line MLE15 23 (Fig. S1B). To further examine the construct in tissues, we electroporated it into embryonic mouse lungs and, after 24 hr culture, observed individual epithelial cells bridging the basement membrane and apical lumen and fluorescing from the 3 fusion proteins with expected distinct distributions (Fig. 1C).

A tricolor reporter mouse labels lysosomes, mitochondria, and microtubules across organs
After validating the kaleidoscope fusion proteins via transient expression in cultured cell lines and lungs, we knocked the construct into the ROSA locus via CRISPR-stimulated homologous recombination in mouse embryonic stem cells (Fig. S1C). To examine the fusion proteins across organs, we activated the resulting ROSA Kaleidoscope allele with a near ubiquitous Cre driver, CMV-Cre 24 . As cell spreading on a 2D culture surface facilitates resolution of subcellular structures, we grew primary cells from dissociated postnatal day 7 (P7) lungs in conditions that most likely favor fibroblasts 25 and compared the kaleidoscope fusion proteins with established markers of lysosomes (LAMP2), mitochondria (TOMM22), and microtubules (beta-Tubulin), with each pair showing remarkable overlaps ( Fig. 2A). The EGFP-alpha-Tubulin was not as filamentous as beta-Tubulin despite the common use of this fusion protein 26,27 , possibly due to extra fusion proteins unincorporated into the microtubule polymers. This was further examined later in the context of cell division. Western blots of whole lungs, in comparison with HEK293T cells transfected with individual fusion proteins, showed consistent patterns except for a lung-specific band possibly due to differential glycosylation of LAMP1 in tissues (Fig. S1D).
The CMV-Cre; ROSA Kaleidoscope mice loaded with multiple fusion proteins were healthy and fertile, and showed typical multi-nucleation in the liver, striation in the skeletal muscles, and a regular array of hair cells in the inner ear (Fig. 2B). Lysosomes were consistently perinuclear but differed in size characteristic for each cell type including, from the largest to the smallest, hepatocytes, outer hair cells, and skeletal muscle cells (Fig. 2B, S2A, S2B). Interestingly, microtubules were aligned perpendicular to skeletal muscle fibers, likely to organize actomyosin bundles, whereas lysosomes and mitochondria showed a longitudinal pattern in the remaining space (Fig. 2B, S2A). Unlike organs dominated by a major cell type, ubiquitous labeling in the lung was uninformative, as its alveolar region has dozens of cell types with intertwined cellular processes (Fig. 2B) -a major rationale to build this genetic reporter for sparse, cell-type-specific labeling, as illustrated below. Nevertheless, the airway epithelium was relatively simple and labeled cells were readily recognizable as ciliated cells with apical microtubule clusters and club cells with apical domes (Fig. S2C, S2D). Comparing across organs, cell types, and ages, we noticed tissue autofluorescence from elastin fibers in the adult lung obscured the kaleidoscope EGFP fusion protein, requiring amplification with a GFP antibody. Relatedly, mKate2 was readily detected in the Cy3 channel, sparing the Cy5 channel for antibody or nuclei staining.

Visualization of microtubule dynamics through cell cycle phases
To further validate the microtubule labeling that was less discrete than the two organelle labelings, we reasoned that mitotic spindle assembly during mitosis would be an informative test case. Reassuringly, in cultured primary lung cells from our reporter mice, the filamentous microtubule network of interphase cells organized into the classic metaphase starbursts in an elevated, rounded cell body (Fig. 3A). To track microtubule dynamics in tissues, we used Sftpc CreER 28 to activate the kaleidoscope reporter in neonatal alveolar type 2 (AT2) cells with ongoing developmental proliferation. We identified microtubule labeling patterns consistent with textbook examples of interphase, prophase, metaphase, anaphase, and telophase, where EGFP-alpha-Tubulin cycled through configurations of a diffuse network, paired foci, paired starbursts, and a bridging bundle (Fig. 3B). Interestingly, while the labeled mitochondria reticulum was largely evenly split between the two separating daughter cells, a single dominant lysosome sometimes formed during mitosis, suggesting delayed assembly or synthesis of new lysosomes that was nevertheless compatible with cell division (Fig. 3B).

Sizes and distributions of lysosomes, mitochondria and microtubules in AT2, AT1 and Cap2 cells
Unlike the small, conventionally shaped AT2 cells, alveolar type 1 (AT1) cells and Cap2 endothelial cells span several hundred microns and contribute to multiple alveoli and capillary segments, respectively 2,5 . More challengingly, both large cells have thin cytoplasm at or below the resolution of optical microscopy and juxtaposed with cytoplasm from other cells, precluding attempts to assign a standard subcellular staining to a given cell type. Accordingly, we used Rtkn2 CreER 29 and newly generated Car4 CreER (Fig. S3A, S3B) to activate the kaleidoscope reporter in sporadic AT1 and Cap2 cells, respectively, with a limiting dose of tamoxifen -a sparse labeling strategy deployed to distinguish adjacent cells of the same type. This revealed for the first time the complete distributions of lysosomes and mitochondria in AT1 and Cap2 cells in the native tissue (Fig. 4). Quantitative imaging showed that AT2 cells had one or two lysosome globules, likely corresponding to lamellar body clusters (verified below with LAMP3 immunostaining) and a rich mitochondrial network, which were both localized to the perinuclear region and respectively occupied around 5 and 15% of the cell volume defined by EGFP-alpha-Tubulin. AT1 cells had close to 50 lysosomal and 100 mitochondrial objects that were smaller or more fragmented and occupied a smaller fraction of the cell volume than those of AT2 cells. Both organelles were distributed throughout the expansive cytoplasm, forming mitochondrial and lysosomal outposts presumably to support active cellular metabolism distant from the nucleus (Fig. 4A, Supplementary Video 1). Cap2 cells had fewer but still abundant lysosomes and mitochondria that tended to accumulate near the nucleus as well as along the microtubule fibers, but infrequently at the cell extremities (Fig. 4A, Supplementary Video 2). The cuboidal AT2 cells were filled with a dense microtubule network, whereas AT1 cells had a mesh of filamentous microtubules, potentially providing mechanical support for their expansive and thin cytoplasm (Fig. 4A). By comparison, Cap2 cells had multiple long microtubule fibers, possibly due to their complex autocellular and intercellular junctions in the 3D capillary tubes (Fig. 4A). The differences in the microtubule density among cell types were reminiscent of those among a cotton ball (AT2 cells), a sieve (AT1 cells), and a fishnet (Cap2 cells) (Fig. S3C).

Live imaging of lysosome movement, fusion, and fission in embryonic lung epithelial progenitors
Expected as a major advantage of fluorescent fusion proteins, the multicolor kaleidoscope reporter in fresh tissues was sufficiently stable for simultaneous live-imaging of lysosomes, mitochondria, and microtubules, and even brighter than fixed tissues (Fig. 5, Supplementary Videos 3-6). Specifically, we activated the reporter with Sox9 CreER in sparse lung epithelial progenitors and explant-cultured such labeled embryonic lungs. Over several hours, lysosomes and mitochondria mostly jittered but occasionally dashed across several microns, with a combined speed averaging 0.16 um/min and 0.14 um/min, respectively (Fig. 5A, Supplementary Video 4, 5). Moreover, the lysosomal puncta fused and fissioned, processes challenging to distinguish from restructuring for the mitochondrial network (Fig. 5A). EGFPalpha-Tubulin was too diffuse under the current imaging condition to identify individual microtubule fibers, but reliably outlined cell nucleus, shape, and location (Supplementary Video 6).

Organelle changes in AT1 cells after Sendai virus infection
After characterizing subcellular structures and dynamics in normal tissues, we applied the kaleidoscope reporter to a Sendai virus injury model, which features AT2-less regions where AT2 cells are depleted as a result of infection and AT1 cells temporarily seal the epithelial barrier before being displaced by invading airway basal-like cells 30 . Interestingly, unlike uninfected lungs and unaffected regions of infected lungs, AT1 cells in AT2-less regions had additional lysosomes but reduced mitochondria, subcellular changes preceding and potentially in preparation for their said displacement by increasing autophagy and reducing energy production (Fig. 6A, Fig. S4). Further supporting cell-type-specific organelle features, AT1 cells lineage-traced from AT2 cells were readily distinguishable from adjacent AT2 cells with their numerous dispersed lysosomes and mitochondria, predicting as drastic a change in subcellular structures as in gene expression during AT2-to-AT1 differentiation (Fig. 6B, Supplementary Video 7).

Yap/Taz mutant lung epithelial cells accelerate lamellar body biogenesis
To further explore the notions that molecular events must be executed by subcellular machinery and that, in the same way that single-cell genomic tools capture molecular states, the kaleidoscope reporter captures subcellular states, we applied our reporter to a cellular analysis of the Yap/Taz mutant lung epithelial progenitors, which was shown via molecular analysis to undergo accelerated AT2 cell differentiation 29 . During normal AT2 cell differentiation, a AT2 cellspecific marker LAMP3 existed in two pools: multiple small puncta in progenitors and developing AT2 cells, and 1-2 large globules in mature AT2 cells, with the second pool overlapping with the kaleidoscope BFP-Lamp1 and gradually increasing in size (Fig. 7). Since large lysosomes were present in early progenitors even before the onset of LAMP3 expression and LAMP3-negative airway cells (Fig. 7A, 7B, S5A), LAMP3 was likely produced as small puncta that subsequently merged with lysosomes to form lamellar body clusters, consistent with them being lysosomal related organelles 31,32 . In the Yap/Taz mutant, both pools of LAMP3, distinguished by an overlap with BFP-Lamp1, were larger in size than those at the same and even later developmental stages (Fig. 7C). This enlargement in both vesicular and clustered LAMP3 could result from over-production of LAMP3 and related proteins due to transcriptional misregulation 29 , overwhelming subcellular machinery for lamellar body production, storage, and secretion. This lysosomal phenotype was consistent with the exaggerated AT2 cell differentiation predicted from molecular profiling 29 ; by comparison, mutant cell mitochondria were not visibly different and mutant cell perimeters fell between AT1 and AT2 cells, possibly due to stretching to compensate for blockage of AT1 cell differentiation and expansion (Fig.  S5B). These diverse cellular phenotypes, akin to differential and unaffected gene expression, highlighted the rich information herein and its use in defining biology.

DISCUSSION
This study represents an initial step to unravel cell-type-specific use of the subcellular machinery in tissues. We generate a Cre-dependent, tricolor fluorescent reporter mouse that simultaneously labels lysosomes, mitochondria, and microtubules across diverse cell types and with single-cell resolution. Applying this tool to the lung with entangled cellular processes of dozens of cell types, we document lysosomal and mitochondrial distributions in the convoluted AT1 and Cap2 cells and upon viral infection; track organelle dynamics in embryonic epithelial progenitors; and reveal accelerated lamellar body biogenesis in a Yap/Taz mutant.
Our study is motivated by the unmet need to extend the extensive knowledge of cell biology in cultured cells to that in native tissues. Such extension is necessary for the same reason that genes often function distinctly across cell types and in ways not recapitulated in culture. Notably, without native matrix, cellular, and geometric environments, cultured cells cannot mimic the full extent of cellular specialization needed for physiology, including endothelial cells in blood-filled vessels, folded ultrathin AT1 cells integrated with neighboring alveolar cells, and their aberrant versions from cumulative developmental deviation due to . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. ; genetic mutations. Such cellular specialization is reflected in not only cell morphology, but also organelle usage such as mitochondrial subpopulations in skeletal muscle cells and lysosomal related organelles such as melanosomes and Weibel-Palade bodies 32,33 . Using the tricolor reporter, we show that AT2 cell lamellar bodies, a type of lysosomal related organelles, form via gradual coalescence of LAMP3 vesicles with lysosomes and this process is accelerated upon Yap/Taz deletion (Fig. 7).
By establishing the feasibility of multicolor imaging of tissue cell biology, our study paves the way for systematic single-cell subcellular analysis, akin to single-cell genomics. First, leveraging fluorescent fusion proteins validated in cultured cells and other safe genomic loci such as Hprt, H11, Tigre, and Col1a1, the next generation of kaleidoscope mice would allow spectral imaging of all known subcellular structures including actin, intermediate filaments, Golgi, ER, endosomes, cell junctions, and matrix adhesions. The resulting kaleidoscopic celltype-specific features represent a modern version of Golgi staining for cell morphology to classify neurons. Second, super-resolution imaging can be used to resolve microtubule dynamics as well as individual mitochondria and lysosomes versus their clusters and, more broadly, to map organelle interactomes in tissues such as mitophage 12 . Third, besides transcriptional regulators such as YAP/TAZ (Fig. 7), these subcellular reporters would enable real time mechanistic studies of direct regulators of cytoskeleton assembly and anchorage; vesicle budding, transport, and fusion; as well as cell junction establishment and remodeling. Their Cre-dependent design will provide definitive evidence for intercellular transfer of organelles such as mitochondria 34 . Last, therapeutic drugs targeting cell biology such as the microtubule stabilizer Taxol can be evaluated for side-effects on all cell types in tissues.

Mice
The following mouse lines were used: Rtkn2 CreER 29 , Sftpc CreER 28 , Sox9 CreER 35 , CMV-Cre 24 , Yap CKO 36 ; Taz CKO 36 .The ROSA Kaleidoscope knock-in allele was generated at the Genetically . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. ; Engineered Mouse Facility at MD Anderson Cancer Center using CRISPR targeting via standard mouse embryonic stem cells (mESCs) transfection. The Kaleidoscope targeting construct was digested with KpnI to linearize the DNA. Linear DNA was then diluted to 2.2 µg/µl and electroporated into 1 x 10 7 G4 (hybrid 129/B6) mESCs. Transfected mESCs were selected for resistance to G418 and puromycin. DNA from individual surviving clones was screened by PCR analysis (see Table 2). Positive clones were then injected separately using standard procedures into blastocysts derived from albino-C57Bl/6N mice to generate male chimeras, which were then mated to black female C57Bl/6N to establish germline transmitted progeny. Presence of the targeted gene was established using PCR analysis of DNA obtained from tail samples ( Table 2).
For embryonic experiments, pregnancies were designated as embryonic day (E) 0.5 on day of plug. Tamoxifen (T5649, Sigma) dissolved in corn oil (C8267, Sigma) was administered via intraperitoneal injection to pregnant mothers at doses and ages in figure legends to induce Cre recombination. Experiments were conducted in both male and female mice and investigators were aware of genotypes at the time of experiments. The mice were housed under conditions of 22°C, 45% humidity, and 12-12 h light-dark cycle. All animal experiments were approved by the Institutional Animal Care and Use at MD Anderson Cancer Center. Every effort was made to process samples in the same tubes as experimental and biological replicates to reduce variation between experiments.

Primary cell culture
P7 CMV-Cre; ROSA Kaleidoscope/+ lungs were removed after perfusion with phosphate buffered saline (PBS), freed of any surrounding tissue and trachea, and minced with forceps into ~1 cm 3 pieces. Tissue fragments were incubated in 1.35 mL PBS plus 150 uL 20 mg/mL Collagenase Type I (Worthington CLS-1 dissolved in PBS) and 15 uL 20 mg/ml DNase I (Worthington D dissolved in PBS) at 37°C for 40 min, with manual mixing halfway and at the end of incubation. Following digestion, tissue was washed with 10% FBS in Leibovitz media (21083-027, Gibco) and spun down. The supernatant was removed and cells incubated in red blood cell lysis buffer (15 mM NH 4 Cl, 12 mM NaHCO 3 , 0.1 uM EDTA, pH8.0) for 3 min before washing as before. Cells were then resuspended in prewarmed RPMI-1640 with 10% FBS, 1% PenStrep and plated on No.1 cover slips in 6-well culture plates. Media was changed daily for 3 days to remove dead cells. On day 4, cells were fixed in 2% PFA for 2 hr, washed with PBS, mounted with Aquapolymount (18606, Polysciences) and imaged.

Embryonic lung explant culture
Time-mated females were harvested at E14.5 and lungs with trachea and glottis were removed from embryos. The Cre-recombined ROSA-Kaleidoscope plasmid and Fast Green FCF dye (MKCD1540, Sigma) were mouth-pipetted through glottis until embryonic lumens were visibly filled. Lungs were electroporated in BioRad Xcell electroporation cuvettes (165-2088, BioRad) at 35 V for 3 rounds of 25 ms with 1s intervals. Lungs were then placed on Nuclepore Track-Etch Membrane filters (10417101, Whatman) and floated on RPMI-1640 media with 10% FBS 1% PenStrep for 24 hr before fixation in 0.5% PFA for 4 hr. After PBS wash, lungs were embedded in optimal cutting temperature compound (OCT; 4583, Tissue-Tek), cryosectioned, and imaged on Deltavision Deconvolution scope.

Live imaging
Time-mated females were injected with 250 ug tamoxifen 2 days prior to sacrifice at E12.5 or E13.5. Embryos were screened for fluorescence and positive lungs were mounted on coverslipbottom plates (P35G-1.5-10-C, Mattek) in a minimal amount of Leibovitz media. Lungs were imaged on a spinning disc confocal (3i Intelligent Imaging Innovations) using a 40x air objective at intervals indicated in figure legends at 37°C with 5% CO 2 and humidified air. Following image acquisition, time-lapse videos were deconvolved using 3i software AutoQuant 3D Blind Deconvolution and converted to tiff files for import to Imaris software.

Section immunofluorescence and confocal imaging
Postnatal and adult lungs were inflation-harvested as described (Yang et al., 2016) with minor modifications. Briefly, mice were sacrificed via intraperitoneal injection of avertin (T48402, Sigma) and perfused through the right ventricle with PBS. The trachea was cannulated and the lung inflated with 0.5% PFA in PBS at 25 cm H 2 O pressure, submersion fixed in 0.5% PFA at room temperature (RT) for 3-6 hr, and washed in PBS at 4°C overnight on a rocker. Section immunostaining was performed following published protocols with minor modifications (Alanis et al., 2014; Chang et al., 2013). Fixed lung lobes were cryoprotected in 20% sucrose in PBS containing 10% OCT at 4°C overnight and then embedded in OCT and frozen at -80°C. Sections were cut at 30 um thickness and blocked in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) in PBS with 0.3% Triton X-100 (PBST) before incubation with primary antibodies diluted in PBST in a humidified chamber at 4°C overnight. Sections were washed with PBS in a coplin jar for 30 min at RT then incubated with donkey secondary antibodies (Jackson ImmunoResearch) diluted in PBST at RT for 1 hr. After another 30 min wash with PBS, sections were mounted with Aquapolymount and imaged in ~20 um z-stacks on a confocal microscope (FV1000V, Olympus) using a 60X oil objective. Liver, inner ear, and muscle tissues were removed following right ventricle perfusion and fixed in 0.5% PFA for 3-6 hr at RT and then cryoprotected and sectioned as above. See Table 3, 4 for a list of antibodies.

Whole mount immunostaining
This was performed following published protocols with minor modifications (Yang et al., 2016). In brief, ~3 mm wide strips from the edge of the left lobes of postnatal or adult lungs or whole lobes of embryonic lungs were blocked with 5% normal donkey serum in PBST for 2-4 hr at RT and then incubated with primary antibodies diluted in PBST overnight at 4°C on a rocker. The strips were then washed with PBS+1%Triton X-100+1%Tween-20 (PBSTT) on a rocker at RT 3 times over 4 hr. Secondary antibodies diluted in PBST were incubated on a rocker overnight at 4°C. On the third day, the strips were washed as above before fixation with 2% PFA in PBS for at least 2 hr on a RT rocker. Finally, the strips were mounted on slides using Aquapolymount with the flattest side facing the coverslip. Z-stack images of 20-40 um thick at 1 um step size were taken from the top of the tissue to obtain an en face view.

Western blot
CMV-Cre; ROSA Kaleidoscope/+ postnatal lungs were removed after PBS perfusion and flash frozen. Tissue was placed in cell lysis buffer (FNN0021, Invitrogen) and protease inhibitor cocktail (78442, Thermo Scientific) and mechanically agitated twice for 45 seconds with chrome steel beads (11079113c, BioSpec Products). Following 10 min incubation on ice, samples were centrifuged at 13,000 rpm for 15 min and supernatant retained. Pierce BCA analysis was performed to determine protein content (23227, ThermoFisher). 40 ug protein was run on a variable percentage acrylamide gel (4561094, BioRad) for 1 hr at 115 V before transferring to protein membrane (IPV00010, Millipore) at 50 V for 2 hr. Membranes were blocked in 5% bovine serum albumin (BSA, A3059, Sigma) in tris-buffered saline (A3059, Sigma) with 0.1% Tween-20 (TBST pH 7.4) at 4°C for 1 hr with constant agitation before incubation with primary antibodies (Table 3) overnight at 4°C with constant agitation. 3-5 short washes in TBST were performed before incubation in 2% BSA in TBST with relevant secondary antibodies (Table 4). Chemiluminescence was imaged on BioRad ChemiDoc gel imager with ~800 ul Immobilon Crescendo Western HRP substrate (WBLUR0100, Millipore). Blots were treated with Restore™ Western Blot Stripping Buffer (21059, ThermoFisher) for 30 min at RT with agitation before blocking and staining as before, up to 2 additional times.

Sendai virus infection
Viral infection was performed as described 30 . Briefly, isoflurane-anesthetized mice were suspended by the upper incisors and treated with a non-lethal dose (~2.1 X10 7 plaque-forming units) Sendai Virus (ATCC #VR-105, RRID:SCR_001672CSCSSCS) in 40 ul PBS or PBS only via oropharyngeal instillation. Lungs were inflation harvested at 14 days post infection, fixed, cryoprotected, and sectioned as described above. Imaris 9.9 (Bitplane) was used for image analysis. Surfaces were created using DAPI (BFP-Lamp1), 488 (EGFP-aTubulin), 555 (mito-mKate2) and 647 (antibody stains) for volume, distance, size, and speed measurements. Identical surface creation parameters were used between experiments. Manual editing for aberrant surfaces was performed prior to statistical analysis. Center Retention Fund, and National Institutes of Health R01HL130129 and R01HL153511 (JC).

AUTHOR CONTRIBUTIONS
VH and JC designed research; VH, AL, and AMGG performed research and analyzed data; JC generated the Car4 CreER mice; VH and JC wrote the paper; all authors read and approved the paper.

COMPETING INTERESTS
The authors declare no competing interests. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Kaleidoscope and cultured for 24 hr. The enlarged cell is truncated by physical sectioning and thus does not reach the airway lumen, as the dimmer cell behind it does. Scale: 10 um. (A) Primary cell culture from dissociated P7 CMV-Cre; ROSA Kaleidoscope/+ lungs, showing colocalization of each fusion protein with markers of their expected subcellular structures: Lamp2 for lysosomes, Tomm20 for mitochondria, beta-Tubulin for microtubules. An escaper of CMV-Cre recombination is outlined with dashes. Scale: 10 um. (B) A survey of cell types and tissues from CMV-Cre; ROSA Kaleidoscope/+ mice at P28, except for the inner ear at P5 to avoid calcification). Lysosomes range in size from 10 to 1 um characteristic of cell types in the decreasing order of hepatocytes, lung alveolar type 2 (AT2) cells, hair cells, skeletal muscle cells, and lung alveolar type 1 (AT1) cells. In the skeletal muscle, microtubules perpendicularly bundle actomyosin fibers, whereas lysosomes and mitochondria align with and fit between actomyosin fibers. Additional lysosomes surround the nuclei (asterisk). Arrow, unrecombined hair cell; n, neuronal cell; s, satellite cell. Scale: 10 um.  Lysosomes and mitochondria are closer to the nucleus in AT2 cells than in AT1 and Cap2 cells, attributable to cell size differences (asterisk, p<0.0001). GFP volume ration is calculated as the percentage of cell volume, approximated by GFP, occupied by lysosomes or mitochondria, and differs among cell types. Statistics is based on ordinary ANOVA with Tukey test.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made    (A) LAMP3 particles shift from non-existent or small, scattered (arrowhead; BFP-) in progenitors and developing AT2 cells (blue and yellow, respectively) to large, overlapping with BFP-Lamp1 (caret; BFP+) in mature AT2 cells (magenta). Unrecombined cells have white outline. More AT2 cells are in the Yap/Taz mutant (dash) and at a more mature stage. 500 ug tamoxifen was given at E13.5 for the control; 2 mg tamoxifen was given at E14.5 for the Yap/Taz mutant. Scale: 10 um. (B) Normal temporal changes in lysosome size from progenitors at E16.5 (500 ug tamoxifen at E12.5) to AT2 cells at E18.5 (500 ug tamoxifen at E14.5) and P7 (300 ug tamoxifen at P1). Scale: 10 um. (C) Quantification of LAMP3 particle volumes over time grouped by overlapping with BFP-Lamp1, showing an earlier increase in volume and a larger end volume in the Yap/Taz mutant. Asterisk, p<0.05; ns, not significant (ordinary ANOVA with Tukey test; data are mean and standard deviation).
plasmid. The Lamp1 sequence is from rat, while the MLS and alpha-Tubulin sequences are from human. See Table 1 for cloning primer sequences and the order of cloning. (B) MLE15 cells transfected with Cre-recombined ROSA-Kaleidoscope to show comparable fluorescence patterns as in HEK 293T cells. Scale: 10 um. (C) Expected PCR genotyping results of ROSA Kaleidoscope littermates. See Table 2

SupFig. 4: Additional examples of labelled AT1 cells upon Sendai virus infection.
Low-magnification view of individual labeled AT1 cells in uninfected lungs and unaffected and AT2-less regions (devoid of LAMP3+ AT2 cells). Boxed regions are magnified. Mitochondrial fluorescence of the AT1 cell in the AT2-less region is not as reduced as in Fig. 5 possibly reflecting an earlier stage of cell displacement. Scale: 10 um.  Fig. 5A, showing BFP-Lamp1. Fig. 5A, showing MLS-mKate2. Fig. 5A, showing EGFP-alpha-Tubulin. Fig. 6B.

SupVideo 7: 3D view of Sftpc CreER lineage-labeled AT1 and AT2 cells, as in
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made      (A) Primary cell culture from dissociated P7 CMV-Cre; ROSA Kaleidoscope/+ lungs, showing colocalization of each fusion protein with markers of their expected subcellular structures: Lamp2 for lysosomes, Tomm20 for mitochondria, beta-Tubulin for microtubules. An escaper of CMV-Cre recombination is outlined with dashes. Scale: 10 um. (B) A survey of cell types and tissues from CMV-Cre; ROSA Kaleidoscope/+ mice at P28, except for the inner ear at P5 to avoid calcification). Lysosomes range in size from 10 to 1 um characteristic of cell types in the decreasing order of hepatocytes, lung alveolar type 2 (AT2) cells, hair cells, skeletal muscle cells, and lung alveolar type 1 (AT1) cells. In the skeletal muscle, microtubules perpendicularly bundle actomyosin fibers, whereas lysosomes and mitochondria align with and fit between actomyosin fibers. Additional lysosomes surround the nuclei (asterisk). Arrow, unrecombined hair cell; n, neuronal cell; s, satellite cell. Scale: 10 um.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 4: Cell-type-specific sizes and distributions of lysosomes and mitochondria in the lung (A) Genetic labeling and Imaris 3D rendering of lysosomes, mitochondria, and microtubules in individual AT2 (Sftpc CreER ; 100 ug tamoxifen at P1 and harvested at P7), AT1 (Rtkn2 CreER ; 50 ug tamoxifen at P1 and harvested at P11), Cap2 (Car4 CreER ; 100 ug tamoxifen at P3 and harvested at P12) cells. n, nucleus of labeled cells based on exclusion of EGFP-alpha-Tubulin; dash, AT1 cell boundary; caret, Cap2 cell edge with few lysosomes and mitochondria. Scale: 10 um. See also Supplementary Video 1, 2. (B) Population and single-cell level measurements with means and standard deviations of lysosomes and mitochondria in genetically labelled AT2, AT1, Cap2 cells. Individual lysosomal or mitochondrial clusters (defined by Imaris segmentation) of AT2 cells are larger than those of AT1 and Cap2 cells (asterisk, p<0.0001). AT1 cells contain more lysosomal and mitochondrial clusters per cell than AT2 and Cap2 cells (asterisk, p<0.001). Lysosomes and mitochondria are closer to the nucleus in AT2 cells than in AT1 and Cap2 cells, attributable to cell size differences (asterisk, p<0.0001). GFP volume ration is calculated as the percentage of cell volume, approximated by GFP, occupied by lysosomes or mitochondria, and differs among cell types. Statistics is based on ordinary ANOVA with Tukey test.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (A) Genetically labeled individual AT1 cells using Rtkn2 CreER in uninfected and 14 days post infection (dpi) with Sendai virus. Compared to uninfected lungs and unaffected regions in the infected lungs, AT1 cells in AT2-less regions, defined by lack of nearby LAMP3, have higher lysosomal fluorescence but lower mitochondrial fluorescence. Scale: 10 um. (B) Genetically labelled AT2 cells using Sftpc CreER in lungs 14 days after Sendai virus infection. Compared to AT2 cells, a lineage-labeled AT1 cell (red mask) has characteristic numerous small lysosomes, supporting reorganization of subcellular structures as AT2 cells differentiate into AT1 cells. AT2 cells are outlined with dashes; AT1 cell organelles are marked by arrowheads. Scale: 10 um. See also Supplementary Video 7.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. ; https://doi.org/10.1101/2023.05.22.541817 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. ; (A) LAMP3 particles shift from non-existent or small, scattered (arrowhead; BFP-) in progenitors and developing AT2 cells (blue and yellow, respectively) to large, overlapping with BFP-Lamp1 (caret; BFP+) in mature AT2 cells (magenta). Unrecombined cells have white outline. More AT2 cells are in the Yap/Taz mutant (dash) and at a more mature stage. 500 ug tamoxifen was given at E13.5 for the control; 2 mg tamoxifen was given at E14.5 for the Yap/Taz mutant. Scale: 10 um. (B) Normal temporal changes in lysosome size from progenitors at E16.5 (500 ug tamoxifen at E12.5) to AT2 cells at E18.5 (500 ug tamoxifen at E14.5) and P7 (300 ug tamoxifen at P1). Scale: 10 um. (C) Quantification of LAMP3 particle volumes over time grouped by overlapping with BFP-Lamp1, showing an earlier increase in volume and a larger end volume in the Yap/Taz mutant. Asterisk, p<0.05; ns, not significant (ordinary ANOVA with Tukey test; data are mean and standard deviation).
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. ; https://doi.org/10.1101/2023.05.22.541817 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. ; https://doi.org/10.1101/2023.05.22.541817 doi: bioRxiv preprint SupFig. 1: Additional details on the design and validation of the Kaleidoscope reporter. (A) Intermediate vector to show cloning sites to concatenate the 3 fusion proteins. The bolded MluI sites used to transfer them to the Ai9 targeting vector to generate the ROSA-Kaleidoscope plasmid. The Lamp1 sequence is from rat, while the MLS and alpha-Tubulin sequences are from human. See Table 1 for cloning primer sequences and the order of cloning. (B) MLE15 cells transfected with Cre-recombined ROSA-Kaleidoscope to show comparable fluorescence patterns as in HEK 293T cells. Scale: 10 um. (C) Expected PCR genotyping results of ROSA Kaleidoscope littermates. See Table 2 for genotyping primer sequences. (D) Western blots of lungs expressing all 3 fusion proteins and HEK 293T cells expressing individual fusion proteins. The second largest band specific to the Kaleidoscope lungs might be due to incomplete glycosylation of LAMP1. Both BFP and mKate2 are tagRFP derivatives and detected by the same antibody.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 23, 2023. ; https://doi.org/10.1101/2023.05.22.541817 doi: bioRxiv preprint